The present invention relates to improved methods for the direct electrochemical synthesis of cysteine and its sulfhydryl analogues as salt-free amino acids, i.e. bases without production of intermediate acid salts.
Cysteine is a sulfhydryl containing amino acid of increasing importance, used in hair wave formulations, nutritional supplements, and as an intermediate in the syntheses of certain pharmaceuticals. L-cysteine is derived from naturally occuring 1-cystine, which is produced by hydrolysis of hair, feathers and other animal products; however, d-cysteine and the racemic optically inactive dl-mixture may also be derived by various methods. Cysteine is known to be unstable in neutral or alkaline media, and is easily oxidized by air to cystine.
Cysteine may be prepared by reduction of cystine, a disulfide, according to the equation: EQU (--S--CH.sub.2 CH(NH.sub.2)CO.sub.2 H) .sub.2 +2H.sup.+ +2e.fwdarw.2 HSCH.sub.2 CH(NH.sub.2)CO.sub.2 H
This reduction has been conducted chemically with reagents such as Na/liquid NH.sub.3, Zn, Al or Sn in aqueous HCl, or solutions of NaBH.sub.4 have been employed. However, these methods lead to impure cysteine contaminated with inorganic by-products which are often difficult or costly to separate, and even minute traces of such impurities may be unacceptable for some uses, like nutritional supplements.
Heretofore, electrochemical reduction of cystine to cysteine was usually conducted in aqueous acid solution in which the cystine was dissolved in aqueous HCl or H.sub.2 SO.sub.4. Rambacher in U.S. Pat. No. 2,907,703 (1959) described the electrochemical reduction of an aqueous suspension of cystine hydrochloride in 2N aqueous HCl solution, using an electrochemical cell containing a cathode of Sn, Cu, Ag, Ni or carbon, in which the anode compartment is separated from the cathode compartment by means of a porous diaphragm. If the cathode is a sheet of Cu or a carbon rod, SnCl.sub.2 is added to the catholyte, and if the cathode is of Ag or Ni, metallic Sn is added to the catholyte. Cysteine as the HCl salt is obtained after prolonged electrolysis. Additional steps are necessary to obtain pure cysteine as the free-base of the amino acid. Thus, with Rambacher's method, in order to prepare cysteine free-base electrochemically, it was necessary to first prepare the acid salt.
Likewise, Wong and Wang, J. Chinese Chem. Soc., 25. 149 (1977) have described the electrochemical reduction of cystine in aqueous HCl solution at stainless steel electrodes in an electrochemical cell fitted with an anion-exchange membrane. The purpose of the anion-exchange membrane is to allow anions, such as chloride ion to pass through the membrane to the anode side of the cell but not allow cations, or the starting material or product through. The electrolysis product, after evaporation of the aqueous electrolyte solution, was cysteine as the HCl salt. The free amino acid cysteine was then prepared by dissolving the cysteine HCl in ethanol, carefully adding aqueous NH.sub.4 OH solution to pH 6.2, and filtering off and drying the free cysteine. Whereas, the electrochemical step gave a 92% yield of cysteine HCl product, the neutralization step gave only an 80% yield of free cysteine. Cysteine is an expensive product, currently about kg, hence losses of cysteine through precipitation steps or otherwise are costly. The Wong and Wang process is impractical on a longer-term production basis, since under these conditions, stainless steel anodes would soon corrode as Cl.sub.2 is evolved at the anode, and moreover Cl.sub.2 or HOCl generated thereby would eventually attack and destroy the kind of anion exchange membrane that was used (Asahi Glass Co., Selemion AMV).
Mizuguchi et al, Bull. Tokyo Inst. Technol. No. 64, 1-6 (1965) conducted electrolyses of cystine in aqueous acid media (HCl or H.sub.2 SO.sub.4 and in aqueous alkaline media (NaOH, Na.sub.2 CO.sub.3 and NH.sub.4 OH), using a porous porcelain diaphragm in a first electrolysis cell to separate anode and cathode compartments. When the aqueous acid solutions were further electrolyzed in a second electrolysis cell containing an ion-exchange resin diaphragm, deacidification to free cysteine was demonstrated to occur in high yield. In alkaline media, Mizuguchi showed that appreciable losses of cystine and cysteine occurred through the porous porcelain diaphragm. Mizuguchi's results with aqueous NH.sub.4 OH solution are particularly pertinent to the present invention. Electrolysis of cystine (12.lg) was conducted at a Pb cathode at a low current density of 25mA/cm.sup.2 using 3M NH.sub.4 OH (about 10% NH.sub.4 OH by weight) with added (NH.sub.4).sub.2 CO.sub.3, in a batch cell containing a porous porcelain diaphragm. After prolonged electrolysis the catholyte solution was evaporated to dryness leaving 9.0g of crude product containing 7.0g of cysteine and 2.0g of cystine. According to the authors, Pb was not detected in the product. Mizuguchi et al concluded at page 6 that alkaline electrolysis provides lower yields of pure cysteine or its salts than acidic electrolysis. Based on actual results, Mizuguchi et al had a calculated yield of cysteine of about 58% and a current efficiency of about 12%, with about 25% of the valuable product and/or valuable starting material lost, presumably through the separator into the anode compartment. A low current efficiency of about 12% under these conditions signifies that most of the cathodic current was used wastefully for H.sub.2 evolution.
Japanese patent No. 58-23450 to Hasaka, first laid open on June 7, 1962 also discloses a process for the electrochemical reduction of cystine to cysteine in aqueous alkaline solutions of ammonia, ammonium carbonate, ammonium chloride, pyridine HCl or piperidine HCl. Hasaka conducted his reaction with a cathode in the form of a low surface area bidimensional plate. Current density was only 10 to 30 mA/cm.sup.2. Like Mizuguchi et al, Hasaka's product yield using alkaline electrolyte was low, ie 75%.
Although the Japanese patent (Hasaka) stresses that low cost metals can now be used with alkaline anolyte which could not be employed with acidic solutions, it has also been discovered that lead cathodes like those of Hasaka are capable of introducing unsafe, toxic levels of lead into the cysteine rendering the product unacceptable particularly as a food grade material for additives, nutritional supplements, an intermediate for synthesis of pharmaceuticals, and other products especially intended for internal as well as external use.
Accordingly there is a need for a more economic, more reliable and efficient method of producing high purity cysteine and its analogues electrochemically from cystine and its corresponding analogues which minimizes losses of costly disulfide feed and sulfhydryl product, does not necessitate additional conductive salts, simplifies the separation of product as the free amino acid from the electrolyte solution, avoids the need for a second deacidification electrolyzer, and provides for a single improved electrolyzer which produces the product at higher current densities, in high yields, current efficiency and conversion.
The present invention provides such improved methods for the electrochemical production of cysteine and its sulfhydryl analogues.